Microscope slide assemblies having a fluid flow-through cell are generally known in the art. For example, the Warner Instruments firm offers microscope chambers for the ml-volume range for micro incubation, for cell microscopy and for cell perfusion. As one of the first manufacturers of such microscope accessories, Warner Instruments has taken on the subject of reversibly assemblable micro flow-through cells. (As used herein, the term reversibly assemblable refers to a product which can be assembled. The principle of the closed microscope chamber can best be seen in the design of flow-through cells for confocal cell microscopy (cf. Warner Instruments Catalog 2002, p. 172). A flat silicon seal in the range of 250 to 1000 μm thick is placed between two cover glasses which are approximately 150 μm thick. One of the cover glasses has openings for the access of fluid. This threefold connection is inserted into a two-piece concentric support and is pressed into a fluid-tight relationship by screwing the two supports into each other. The upper cover plate of the concentric support, for which there is room on the microscope work plate, contains the fluid inlets.
This method permits the use of at least one disposable cover glass and of a second, apertured cover glass or a slide, especially adapted to the design of the flow-through channel. The channel geometry is limited to that which is technically feasible in the structuring of the flat silicon seal, i.e., the channels are only accurate to one millimeter and can be made no thinner than 250 μm. A further drawback is that the channels can only have rough contours and cannot be fabricated with internal channel barriers, such as flow guides or flow splitters. Further, the channels cannot be adjusted accurate to microns to structures of one or both cover glasses, and numerous applications with electrically functionalized surfaces simply cannot be performed.
The flat silicon seal is relatively costly, has a complicated chamber assembly that can easily be maladjusted in installation and is hard to keep clean. Owing to the restrictions in geometry, only relatively large-volume channels can be realized, and therefore Warner Instruments only offers the system for the ml-volume range.
The firm of Ibidi GmbH offers a new generation of μ-slides, which are suitable as microscope supports of synthetic material for common and high-resolution microscopy methods, such as for example DIC, phase contrast, fluorescence and transmitted light. This slide-based technique is directed to use as a disposable product on the microscope, designed to work without connection to components of automated external fluidics.
Another product known in the art is the so-called μ-slide I, which is offered as a flow-chamber system for cell culture and in vitro cell microscopy. This system is an irreversibly connected channel system, consisting of two planes of synthetic material—the embossed base and a perforated cover. The μ-slide I consists of a 100-μl channel, which on both sides ends in a 2-ml fluid reservoir. The 2-ml reservoirs in practice form a cell culture dish which is directly connected to the simple flow-through system for microscopic observation of appropriate objects on the inverse microscope. Objects are flowed into the channel, and flow parameters and fluid composition are not influenced, as is possible with the connection of external pumps and valves.
A second product defining the related art is Ibidi GmbH's μ-slide V. Designed according to the principle of the μ-slide I, this is also a flow-chamber system for simultaneous protein analysis in five channels running parallel through the microscope image field and each holding 17 μl. The fluid connections to the five channels each form an inlet and outlet reservoir and, here again, there is no possibility of influencing flow parameters by external fluidics.
A third product defining the known art is the micro array μ-slide of Ibidi GmbH. This is a two-piece flow-chamber system, still open before use for work on the microscope, consisting of two parts of synthetic material, the embossed base and the one-time self-adhesive unembossed cover.
The μ-slide is primarily used for rapid micro array analysis by in-situ hybridization in a flow-through mode. For this purpose, the micro array is produced by using a spotting technique in the chamber region of the initially open base, then the cover is irreversibly applied and flow-through hybridization can take place.
After hybridization of the array, evaluation under the microscope takes place and then the slide is discarded. The fluid circuit is characterized by an inlet opening for conventional disposable plastic pipette tips, the 10 to 400 μl-reaction chamber and a collecting reservoir at the outlet of the reaction chamber.
Corresponding to its principle of use as a disposable diagnostic system, Ibidi microscope cells are low-cost disposable items and, as such, are suitable for a very limited area of application in terms of method. Flow-through analyses, test procedures or handling techniques that require continuous flow-through operation cannot be performed with the Ibidi systems μ-slide.
Several firms have recently offered flow-through cells for microscopy in the μl-volume range; these systems are based on glass, ceramic, synthetic material or silicon and permit inclusion of the growing field of microsystem technology, biotechnology and nanotechnology.
Ippei Inoue has developed a cell culture system on chips—the glass array slide used as a micro flow-through cell for work under the optical microscope [Ippei, Inoue, et al.; On chip culture system for observation of isolated individual cells; Lab on a Chip, 2001 1, 50-55]. The system was a glass slide in which microcavities arranged array-like are etched. The micro flow-through cell is produced by application of a covering plate on the array slide. If this micro flow-through cell is placed on the work plate of an inverse microscope and connected to an external fluid supply, the fluidic process and microscopic observation can be performed simultaneously.
The microcavities are produced by isotropic etching of the glass. The microcavities introduced measure in the range of 20 to 70 μm in diameter and about 5 to 30 μm in depth. After etching, the surface of the glass was functionalized on a nano scale by attachment of amino groups and biotin. Commercially available chemicals, which were applied to the glass by immersion, tempering and wash techniques can be used for this purpose. The microcavities are then filled with cells and sealed with a semi-permeable cellulose membrane functionalized with streptavidin. Thus, in this system, the biotin-streptavidin connection provides a reliable sealing mechanism between the microcavities filled with cells and the membrane. Accordingly, this closure is effected without cementing and without elastic sealing.
This is an example of the use of strong biotin-streptavidin bonding for solving an assembly problem in biotechnology. If this cell-based array slide is produced on the slide, a micro scale single-channel glass cover is placed on top and nutrient solution can be supplied from outside. The replacement of nutrient medium in each micro cavity covered with a cellulose membrane takes place by diffusion through the covering membrane. The disadvantage of this biotechnological assembly method is that a biochemically realizable biotin/avidin contact is permanently connected.
Another example of the realization of microscope cells is a method proposed by the GeSiM firm in patent PCT/DE 01/03324. Here, photolithographic structuring techniques and SMD assembly technology are linked in such a way that base glass and cover glass of the microscope cell may be positioned to one another accurate to microns, and permanently cemented together. This technology is very interesting for reusable products, but clearly too costly for disposable products.